Mutants of a pseudomonas glumae butynol esterase

ABSTRACT

The invention relates to novel proteins having esterase activity, to mutants thereof, to nucleic acid sequences coding therefor, to expression cassettes, vectors and recombinant microorganisms; to methods for preparing said proteins and to the use thereof for enzymic, in particular enantioselective enzymic, ester hydrolysis or transesterification of organic esters.

RELATED APPLICATIONS

This application is a national stage application (under 35 U.S.C. 371)of PCT/EP2007/056367, filed Jun. 26, 2007, which claims benefit ofEuropean application 06013236.2, filed Jun. 27, 2006.

SUBMISSION OF SEQUENCE LISTING

The Sequence Listing associated with this application is filed inelectronic format via EFS-Web and hereby incorporated by reference intothe specification in its entirety. The name of the text file containingthe Sequence Listing is Sequence_Listing_(—)13111_(—)00113. The size ofthe text file is 33 KB, and the text file was created on Jan. 24, 2011.

The invention relates to novel proteins having esterase activity, tofunctional equivalents and mutants thereof, to nucleic acid sequencescoding therefor, to expression cassettes, vectors and recombinantmicroorganisms; to methods for preparing said proteins and to the usethereof for enzymic, in particular enantioselective enzymic, esterhydrolysis or transesterification of organic esters.

BACKGROUND OF THE INVENTION

Esterases and lipases are hydrolases which can be employed in industrialprocesses for synthesizing optically active organic compounds and whichare characterized by high substrate specificity. Through a mechanismsimilar to that of serine proteases, they can transfer acyl groups ontonucleophiles such as, for example, carbonyl groups or hydrolyticallycleave ester bonds. Esterases, lipases and serine proteases share thecatalytic triad, a sequence motif consisting of the amino acids Ser, Hisand Asp, where the carbonyl carbon atom is subject to nucleophilicattack by the active Ser, which, with participation of the other twoamino acids, leads to a charge distribution. Esterases and lipases mayalso transfer acyl groups onto other nucleophiles, such as thioetherthio groups or activated amines.

Lipases hydrolyze long-chain glycerol esters and are characterized bysurface activation, i.e. the active site becomes accessible only in thepresence of the lipid substrate. Lipases are stable in nonaqueousorganic solvents and are employed in numerous industrial processes forkinetic racemate resolution, i.e. one enantiomer is convertedsubstantially faster than the other. Said enantiomer can be subsequentlyobtained from the reaction solution owing to different physical andchemical properties.

Nakamura (Nakamura, K. et al., Tetrahedron; Asymmetry 9, (1999),4429-4439) describes the racemate resolution of 1-alkyn-3-ol bytransesterification in hydrophobic solvents with the aid of commerciallyavailable lipases (Amano AK, AH and PS; Amano Pharmaceuticals Co. Ltd.).In this reaction, enantioselectivity increases with the chain length ofthe acyl donor and sterically large residues (chloroacetate, vinylbenzoate) have an adverse effect on the reaction. Yang (Yang, H. et al.,J. Org. Chem. 64, (1999), 1709-1712) describes the enantioselectivepreparation of optically active acids by transesterification with vinylesters using lipase B from Candida antarctica as catalyst. In this case,ethyl esters lead to a distinctly lower reaction rate and selectivity. Alipase isolated from Burkholderia plantarii (Pseudomonas plantarii orglumae) DSM 6535 is employed for enantioselective acylation of racemicamities with the aid of ethyl methoxyacetate (Balkenhohl, F. et al., J.prakt. Chem. 339, (1997), 381-384).

Esterases enantioselectively catalyze the formation and breaking ofester bonds (forward and reverse reaction). Preference is given to usingvinyl esters in the transesterification for obtaining optically activealcohols, since the alcohol function of the ester is no longer availableafter the conversion due to tautomerization to the aldehyde or ketoneand thus the reverse reaction can be avoided. In contrast to lipases,esterases are not surface-activated and also convert organic compoundsof relatively short chain length. Esterases of different substratespecificity have been isolated from various organisms.

Thus the esterase from Pseudocardia thermophila FERM-BP-6275 is used forhydrolyzing optically active chromanacetic esters (EP-A-0 892 044).

An esterase from Bacillus acidocaldarius hydrolyzes with lowenantioselectivity esters from a narrow range of substrates (Manco, G.et al., Biochem. J. 332, (1998), 203-212).

Acylase 1 from Aspergillus is used for obtaining secondary alcohols bytransesterification with vinyl esters in organic nonpolar solvents, itbeing preferred to convert secondary alcohols having short side chains(Faraldos, J. et al., Synlett 4, (1997), 367-370). From Pseudomonasfluorescens DSM 50 106 a membrane-bound lactone-specific esterase hasbeen cloned (Khalameyzer, V. et al., Appl. and Environ. Microbial.65(2), (1999), 477-482), and from the E. coli Q mutant an acetylesterasehas been cloned (Peist, R. et al., J. Bacteriol. 179, (1997),7679-7686). However, enantioselectivity and substrate specificity ofthese two esterases have not been studied in more detail. Rhodococcussp. NCBM 11216 expresses 4 esterases, RR1 to RR4, which have differentspecificity. For the ester synthesis from naphthol and an acid, RR1 andRR2 prefer acids with short carbon chains, while RR3 and RR4specifically convert acids having relatively long carbon chains andsterically relatively large residues (Gudelj, M. et al., J. Mol. Cat. B,Enzymatic 5, (1998), 261-266).

However, until recently esterases which possess a broad substrate range,have high enantioselectivity and can be employed in industrial processeshave not been available for preparing small organic molecules such asoptically active alcohols, acids or esters with short carbon chains.

WO-A-02/18560, for the first time, has described useful proteins havingesterase activity, referred to as esterases therein, which are capableof enantioselective hydrolysis of a wide range of optically activeesters. More specifically, it has described a protein comprising 510amino acids (SEQ ID NO: 2) and the coding sequence thereof (SEQ ID NO:1).

It is the object of the present invention to provide further optionallyactivity-optimized esterases which have at least one of theabovementioned properties.

SUMMARY OF THE INVENTION

Said object was surprisingly achieved by providing a protein havingesterase activity or a functionally equivalent protein thereof, whereinsaid protein has a polypeptide chain with a total length of less than510 amino acids and wherein said chain comprises at least one partialamino acid sequence according to SEQ ID NO: 3.

DESCRIPTION OF THE FIGURES

FIG. 1 depicts a sequence alignment of a partial amino acid sequence ofbutynol I esterase to a partial sequence of a lactone-specific esterasefrom Pseudomonas fluorescens. Query: partial sequence of the clone ofthe invention, LU2898 (SEQ ID NO: 25). Sbjct: partial sequence of the P.fluorescens enzyme (Accession No.: 087637; SEQ ID NO: 26).

FIG. 2 illustrates the cloning chart for LU2898.

FIG. 3 depicts a comparison of the butynol esterase gene from LU2898with the ERGO database. Regions of high homology are in dark. The textcolumns on the right-hand side are annotations from ERGO and indicatethe corresponding e values; the e value is the probability of thesequence homology being based on chance. The position of the PstIcleavage site in the butynol esterase gene is indicated. For comparison,the amino acid sequence of the non-recombinant enzyme from Ps. glumaeLU2023.

FIG. 4 illustrates the enzymic activity of an esterase of the inventionhaving a sequence of 335 amino acids in length (from clone LU11147) andof activity-enhanced mutants derived therefrom.

DETAILED DESCRIPTION OF THE INVENTION 1. Preferred Embodiments

A first subject matter of the invention relates to a protein havingesterase activity or a functionally equivalent protein thereof, whereinsaid protein has a polypeptide chain with a total length of less than510 amino acids and wherein said chain comprises at least one partialamino acid sequence according to SEQ ID NO: 3, said protein preferablypossessing an esterase activity which can be characterized by way ofcleavage of the ester but-3-yn-2-yl butyrate as reference substrate.

Proteins of the invention may in particular additionally comprise atleast one further partial amino acid sequence according to SEQ ID NO: 4,5 or 6.

Said partial amino acid sequences according to SEQ ID NO: 3, 4, 5 or 6are defined as follows (indicated in each case in the one-letter code ofamino acids, with the first amino acid in each case corresponding to theparticular amino-terminal end):

a) FIETLGLERPVLVGHSLGGAIALAVGLDYPER, (SEQ ID NO: 3) b) IALIAPLTHTETEP,(SEQ ID NO: 4) c) GGGMMGLRPEAFYAASSDLV (SEQ ID NO: 5) d) AIDAIFAPEPV(SEQ ID NO: 6)

The proteins of the invention comprise in particular a polypeptide chainhaving less than 450, such as from 300 to 445 for example, or less than350 amino acids, such as in particular from 345 to 300, in particularfrom about 330 to 340, especially 335, amino acids in total length.

Particular mention may be made of a protein comprising an amino acidsequence according to SEQ ID NO: 8.

The invention further relates to activity-enhanced mutants of thesenovel truncated esterases and also to similarly prepared mutants of theesterases disclosed in WO-A-02/18560 and comprising about 510 aminoacids and functional equivalents thereof.

The invention relates in particular to esterase mutants having at leastone functional mutation in any of the amino acid sequence regions 12-20,185-195 and 258 to 268 of SEQ ID NO:2 or 8 and especially at least onefunctional mutation in any of the amino acid sequence positions 16, 190and 263 of SEQ ID NO:2 or 8.

Nonlimiting examples of such mutations are the following amino acidsubstitutions: Leu16Pro, Ile190Thr, Ile190Arg and Ile263Val, eitheralone or in any combination.

The proteins of the invention are moreover a polypeptide chain having acalculated molecular weight of about 60 kDa or less, such as 56 kDa orless or 55.5 kDa or less, for example. For example, the truncatedbutynol esterases with fewer than 510 amino acids may have a molecularweight in the range from about 56 kDa to 20 kDa, for example from about55.5 to 30 kDa or from 55.5 to 35 kDa or from 55.5 to 40 kDa or from 40to 30 kDa, or from 55.5 to 45 kDa or from 38 to 34 kDa or from 36.5 to35.5 kDa, for example.

Mutants of proteins according to SEQ ID NO: 2 preferably have calculatedmolecular weights in the range from about 60 to 40 kDa, or from 56 to 50kDa or from 55.5 to 54 kDa.

Mutants of proteins according to SEQ ID NO: 8 preferably have calculatedmolecular weights in the range from about 38 to 34 kDa, from 37 to 35kDa or from 36.5 to 35.5 kDa.

Preferred proteins can be obtained from Pseudomonas glumae (alsoreferred to as Burkholderia plantarii) LU2023 with deposition number DSM13176 (deposited with the DSMZ on Dec. 2, 1999) and, if appropriate,subsequent mutation.

The inventive proteins having esterase activity, functional equivalentsand mutants moreover catalyze at least one of the following reactions:

-   -   a) enantioselective hydrolysis of optically active esters of the        formula I        R¹—COO—R²  (I),    -   in which    -   R¹ is a straight-chain or branched, optionally mono- or        polysubstituted C₁-C₁₀-alkyl, C₂-C₁₀-alkenyl, C₂-C₁₀-alkynyl,        and R² is a straight-chain or branched, optionally mono- or        polysubstituted C₁-C₁₀-alkyl, C₂-C₁₀-alkenyl, C₂-C₁₀-alkynyl,        C₇-C₁₅-aralkyl or a mono- or polynuclear, optionally mono- or        polysubstituted aromatic radical,    -   R¹ and/or R² comprise at least one asymmetric carbon; and    -   b) enantioselective transesterification of an ester of the        formula I with an optically active alcohol of the formula II        R²—OH  (II),        -   in which R² has one of the above meanings and optionally has            at least one asymmetric carbon.

Examples of suitable C₁-C₁₀-alkyl radicals, which may be mentioned, arestraight-chain or branched radicals with from 1 to 10 carbons, such asmethyl, ethyl, isopropyl or n-propyl, n-, iso-, sec- or tert-butyl,n-pentyl or isopentyl; moreover n-hexyl, n-heptyl, n-octyl, n-nonyl,n-decyl, and also the singly or multiply branched analogs thereof.

Examples of suitable C₂-C₁₀-alkenyl radicals are themono-polyunsaturated analogs of the abovementioned alkyl radicals withfrom 2 to 10 carbons, which analogs preferably have one or twocarbon-carbon double bonds which may be in any position of the carbonchain.

Examples of suitable C₂-C₁₀-alkynyl radicals are the mono- orpolyunsaturated analogs of the abovementioned alkyl radicals with from 2to 10 carbons, which analogs preferably have one or two carbon-carbontriple bonds which may be in any position of the carbon chain.

C₇-C₁₅-Aralkyl is preferably phenyl-C₁-C₅-alkyl or naphthyl-C₁-C₅-alkyl.

Examples of a mononuclear or polynuclear, optionally mono- orpolysubstituted aromatic radical, which may be mentioned, are phenyl andnaphthyl that are substituted with 1, 2 or 3 identical or differentsubstituents selected from among C₁-C₅-alkyl such as methyl, ethyl,isopropyl or n-propyl, n-, iso-, sec- or tert-butyl, n-pentyl orisopentyl; hydroxy, mercapto, amino, nitro or halo such as F, Br, Cl.

The ester of the formula I is derived, for example, from straight-chainor branched, optionally mono- or polyunsaturated, optionally substitutedC₁-C₁₁-monocarboxylic acids. Mention may be made by way of example of:saturated acids such as formic acid, acetic acid, propionic acid andn-butyric acid and i-butyric acid, n-valeric acid and isovaleric acid,caproic acid, enanthoic acid, caprylic acid, pelargonic acid, capricacid, undecanoic acid; monounsaturated acids such as acrylic acid,crotonic acid; and diunsaturated acids such as sorbic acid. If the acidscomprise double bonds, then the latter may be both in cis and in transform.

The invention furthermore relates to polynucleotides coding for aprotein, a functional equivalent or a mutant as defined above, and tofunctional equivalents of said polynucleotides, polynucleotidescomplementary thereto and to the nucleic acid sequences hybridizabletherewith.

The invention relates in particular to those polynucleotides comprisinga nucleotide sequence of at least 30 consecutive nucleotide residues ina nucleic acid sequence according to SEQ ID NO: 7.

The invention moreover relates to a polynucleotide wherein the codon inthe region corresponding to amino acid position 263 of SEQ ID NO: 8 isselected from among GTT and GTC.

The invention further relates to expression cassettes comprising atleast one polynucleotide as defined above and operatively linked to atleast one regulatory nucleic acid sequence.

The invention further relates to recombinant vectors for transforming aeukaryotic or prokaryotic host, comprising a polynucleotide as definedabove or an expression cassette as defined above.

The invention further relates to a process for preparing a protein asdefined above, which comprises culturing a microorganism which producessaid protein endogenously or a microorganism transformed with a vectoras defined above and isolating said protein from the culture.

Suitable processes in this connection use the microorganism Pseudomonasglumae (Burkholderia plantarii) LU2023 with deposition number DSM 13176or a microorganism derived therefrom.

The invention also relates to a protein as defined above and tofunctional equivalents or mutants thereof, obtainable according to aprocess as defined above.

The invention furthermore relates to Pseudomonas glumae (Burkholderiaplantarii) LU2023 with deposition number DSM 13176 and to variants andmutants thereof.

The invention moreover relates to microorganisms carrying a vector asdefined above.

The invention furthermore relates to a process for enantioselectiveester hydrolysis using a protein (including functional equivalents andmutants) as defined above, which process comprises

-   -   a) contacting said protein with a stereoisomer mixture of an        optically active ester of the formula I; and    -   b) obtaining the optically active compounds produced by        stereoselective hydrolysis of any of said stereoisomers and/or        the nonhydrolyzed ester enantiomer from the reaction medium.

The invention also relates to a process for enantioselectivetransesterification, which comprises

-   -   a) contacting a stereoisomer mixture of an optically active        alcohol of the formula II with an ester of the formula I in the        presence of a protein (including functional equivalents and        mutants) as defined above and obtaining the unreacted alcohol        stereoisomer from the reaction medium; or    -   b) contacting a stereoisomer mixture of an optically active        ester of the formula I with an alcohol of the formula II in the        presence of a protein (including functional equivalents and        mutants) as defined above and obtaining a stereoisomer of the        optically active alcohol comprised in said ester from the        reaction medium.

According to a particular variant of this process, the acylating agentfor an optically active alcohol, which is used for transesterification,is a vinyl ester.

The invention in particular relates to a process as defined above,wherein the reaction medium used is an organic solvent.

2. Explanation of General Terms

An “esterase” or “butynol esterase” or “butynol I esterase” is an enzymewhich catalyzes at least one of the enzymic conversions indicatedherein, in particular at least cleavage of a reference butynol ester ofa carboxylic acid, in particular of butyric acid, such as for example abutynol butyrate such as but-3yn-2yl butyrate.

A sequence “derived” from a specifically disclosed sequence or“homologous” thereto, for example a derived amino acid sequence ornucleic acid sequence, means according to the invention, unlessindicated otherwise, a sequence which is at least 80% or at least 90%,in particular 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99%, identicalto the starting sequence.

“Identity” between two nucleic acids means identity of the nucleotidesover the entire nucleic acid length in each case, in particular theidentity calculated by comparison with the aid of the Vector NTI Suite7.1 software from Informax (USA) using the Clustal method (Higgins D G,Sharp P M. Fast and sensitive multiple sequence alignments on amicrocomputer. Comput Appl. Biosci. 1989 April; 5(2):151-1), setting thefollowing parameters:

Multiple alignment parameter: Gap opening penalty 10  Gap extensionpenalty 10  Gap separation penalty range 8 Gap separation penalty off %identity for alignment delay 40  Residue specific gaps off Hydrophilicresidue gap off Transition weighing 0 Pairwise alignment parameter: FASTalgorithm on K-tuple size 1 Gap penalty 3 Window size 5 Number of bestdiagonals 5

3. Further Embodiments of the Invention

3.1 Proteins According to the Invention

The present invention is not limited to the specifically disclosedproteins and enzymes having esterase activity but rather extends also tofunctional equivalents thereof.

For the purposes of the present invention, “functional equivalents” oranalogs of the specifically disclosed enzymes are polypeptides whichdiffer from said enzymes and which furthermore possess the desiredbiological activity such as hydrolytic activity, for example.

“Functional equivalents”, for example, mean enzymes whose activity inthe esterase activity assay used is at least 1%, such as for example atleast 10% or 20%, such as for example at least 50% or 75% or 90%, higheror lower than the activity of an enzyme comprising an amino acidsequence according to SEQ ID NO: 8. Moreover, functional equivalents arepreferably stable between pH 4 to 10, with their optimal pHadvantageously being in a range from pH 5 to 9, such as 6 to 8, forexample, and their optimal temperature being in a range from 15° C. to80° C. or 20° C. to 70° C.

The esterase activity may be detected with the aid of various knownassays. Without being limited thereto, an assay may be mentioned using areference substrate such as, for example, a butynol butyrate such asbut-3yn-2yl butyrate, under standardized conditions (such as e.g. 20 mMsubstrate, 10 mM phosphate buffer, pH 7.4, T=20° C.).

“Functional equivalents” mean according to the invention in particularalso “mutants” which have a different amino acid than the specificallymentioned one in at least one sequence position of the abovementionedamino acid sequences, but which nevertheless possess one of theabovementioned biological activities. “Functional equivalents” thuscomprise the mutants obtainable by one or more amino acid additions,substitutions, deletions and/or inversions, it being possible for saidmodifications to occur in any sequence position as long as they lead toa mutant having the property profile of the invention. Functionalequivalence exists in particular also when the reactivity patternsbetween mutant and unmodified polypeptide agree qualitatively, i.e., forexample, identical substrates are converted at different rates. Examplesof suitable amino acid substitutions are summarized in the followingtable:

Original residue Examples of substitution Ala Ser Arg Lys Asn Gln; HisAsp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn; Gln Ile Leu; Val LeuIle; Val Lys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr ThrSer Trp Tyr Tyr Trp; Phe Val Ile; Leu

Specific examples of individual sequence regions suitable for carryingout mutations according to the invention have already been definedabove, namely: the amino acid sequence regions 12-20, 185-195 and 258 to268 from SEQ ID NO:2 or 8, in particular the amino acid sequencepositions 16, 190 and 263 of SEQ ID NO:2 or 8.

Specific examples of suitable amino acid substitutions are Leu16Pro,Ile190Thr, Ile190Arg and Ile263Val. Further modifications thereof may beprovided readily by the skilled worker in knowledge of the teaching ofthe present invention.

“Functional equivalents” in the above sense are also “precursors” of thedescribed polypeptides and also “functional derivatives” and “salts” ofsaid polypeptides.

“Precursors” are in that case natural or synthetic precursors of thepolypeptides with or without the desired biological activity.

The expression “salts” means salts of carboxyl groups as well as acidaddition salts of amino groups of the protein molecules according to theinvention. Salts of carboxyl groups can be produced in a manner knownper se and comprise inorganic salts, for example sodium, calcium,ammonium, iron and zinc salts, and salts with organic bases, for exampleamines, such as triethanolamine, arginine, lysine, piperidine and thelike. Acid addition salts, for example salts with mineral acids, such ashydrochloric acid or sulfuric acid and salts with organic acids, such asacetic acid and oxalic acid, are also covered by the invention.

“Functional derivatives” of polypeptides according to the invention canalso be produced on functional amino acid side groups or at theirN-terminal or C-terminal end using known techniques. Such derivativescomprise for example aliphatic esters of carboxylic acid groups, amidesof carboxylic acid groups, obtainable by reaction with ammonia or with aprimary or secondary amine; N-acyl derivatives of free amino groups,produced by reaction with acyl groups; or O-acyl derivatives of freehydroxy groups, produced by reaction with acyl groups.

“Functional equivalents” naturally also comprise polypeptides that canbe obtained from other organisms, as well as naturally occurringvariants. For example, areas of homologous sequence regions can beestablished by sequence comparison, and equivalent enzymes can bedetermined on the basis of the specific parameters of the invention.

“Functional equivalents” also comprise fragments, preferably individualdomains or sequence motifs, of the polypeptides according to theinvention, which for example display the desired biological function.

“Functional equivalents” are, moreover, fusion proteins, which have oneof the polypeptide sequences stated above or functional equivalentsderived therefrom and at least one further, functionally different,heterologous sequence in functional N-terminal or C-terminal linkage(i.e. without substantial mutual functional impairment of the fusionprotein parts). Nonlimiting examples of such heterologous sequences aree.g. signal peptides, histidine anchors or enzymes.

“Functional equivalents” that are also included according to theinvention are homologs of the specifically disclosed proteins. Thesepossess at least 60%, preferably at least 75% in particular at least85%, e.g. 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%, homology with oneof the specifically disclosed amino acid sequences, calculated accordingto the algorithm of Pearson and Lipman, Proc. Natl. Acad, Sci. (USA)85(8), 1988, 2444-2448. A percentage homology of a homologouspolypeptide according to the invention means in particular thepercentage identity of the amino acid residues based on the total lengthof one of the amino acid sequences specifically described herein.

In the case of a possible protein glycosylation, “functionalequivalents” according to the invention comprise proteins of the typedesignated above in deglycosylated or glycosylated form as well asmodified forms that can be obtained by altering the glycosylationpattern.

Homologs of the proteins or polypeptides according to the invention canbe produced by mutagenesis, e.g. by point mutation, extension ortruncation of the protein.

Homologs of the proteins according to the invention can be identified byscreening combinatorial libraries of mutants, for example truncationmutants. For example, a variegated library of protein variants can beproduced by combinatorial mutagenesis at the nucleic acid level, e.g. byenzymatic ligation of a mixture of synthetic oligonucleotides. There area great many methods that can be used for the production of libraries ofpotential homologs from a degenerated oligonucleotide sequence. Chemicalsynthesis of a degenerated gene sequence can be carried out in anautomatic DNA synthesizer, and the synthetic gene can then be ligatedinto a suitable expression vector. The use of a degenerated set of genesmakes it possible to supply all sequences in a mixture, which code forthe desired set of potential protein sequences. Methods of synthesis ofdegenerated oligonucleotides are known to a person skilled in the art(e.g. Narang, S. A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu.Rev. Biochem. 53:323; Itakura et al., (1984) Science 198:1056; Ike etal. (1983) Nucleic Acids Res. 11:477).

In the prior art, several techniques are known for the screening of geneproducts of combinatorial libraries, which have been produced by pointmutations or truncation, and for the screening of cDNA libraries forgene products with a selected property. These techniques can be adaptedfor the rapid screening of the gene libraries that have been produced bycombinatorial mutagenesis of homologs according to the invention. Thetechniques most frequently used for the screening of large genelibraries, which are based on a high-throughput analysis, comprisecloning of the gene library in expression vectors that can bereplicated, transformation of suitable cells with the resultant vectorlibrary and expression of the combinatorial genes in conditions in whichdetection of the desired activity facilitates isolation of the vectorthat codes for the gene whose product was detected. Recursive EnsembleMutagenesis (REM), a technique that increases the frequency offunctional mutants in the libraries, can be used in combination with thescreening assays, in order to identify homologs (Arkin and Yourvan(1992) PNAS 89:7811-7815; Delgrave et al. (1993) Protein Engineering6(3):327-331).

Further examples of functional equivalents of the esterases according tothe invention comprise, for example, at least one partial sequencederived from SEQ ID NO: 3, 4, 5 or 6, with one or more amino acidshaving been substituted, deleted, inverted or added compared to thespecifically indicated partial sequence and with the esterase activitydiffering from the esterase activity of the native protein by no morethan ±90% or ±50%, preferably by no more than ±30%.

The esterases according to the invention are obtainable in particularfrom Pseudomonas glumae LU2023, deposition number DSM 13176. Furtherstrain variants are available, for example starting from Pseudomonasglumae LU8093, by selection, for example by way of culturing on minimalmedium plates with ethylphenyl acetate as the sole carbon source.

3.2 Coding Nucleic Acid Sequences

The invention also relates to nucleic acid sequences that code for anenzyme with esterase activity. Nucleic acid sequences comprising asequence according to SEQ ID NO: 7; or a nucleic acid sequence derivedfrom the amino acid sequence according to SEQ ID NO.: 8 are preferred.

All the nucleic acid sequences mentioned herein (single-stranded anddouble-stranded DNA and RNA sequences, for example cDNA and mRNA) can beproduced in a manner known per se by chemical synthesis from thenucleotide building blocks, e.g. by fragment condensation of individualoverlapping, complementary nucleic acid building blocks of the doublehelix. Chemical synthesis of oligonucleotides can, for example, beperformed in a known way, by the phosphoamidite method (Voet, Voet, 2ndedition, Wiley Press, New York, pages 896-897). The accumulation ofsynthetic oligonucleotides and filling of gaps by means of the Klenowfragment of DNA polymerase and ligation reactions as well as generalcloning techniques are described in Sambrook et al. (1989), MolecularCloning: A laboratory manual, Cold Spring Harbor Laboratory Press.

The invention also relates to nucleic acid sequences (single-strandedand double-stranded DNA and RNA sequences, e.g. cDNA and mRNA), codingfor any of the above polypeptides and their functional equivalents,which can be obtained for example using artificial nucleotide analogs.

The invention relates both to isolated nucleic acid molecules, whichcode for polypeptides or proteins according to the invention orbiologically active segments thereof, and to nucleic acid fragments,which can be used for example as hybridization probes or primers foridentifying or amplifying coding nucleic acids according to theinvention.

The nucleic acid molecules according to the invention can in additioncomprise untranslated sequences from the 3′ and/or 5′ end of the codinggenetic region.

The invention further relates to the nucleic acid molecules that arecomplementary to the specifically described nucleotide sequences or asegment thereof.

The nucleotide sequences according to the invention make possible theproduction of probes and primers that can be used for the identificationand/or cloning of homologous sequences in other cell types andorganisms. Such probes and primers generally comprise a nucleotidesequence region which hybridizes under “stringent” conditions (seebelow) to at least about 12, preferably at least about 25, for exampleabout 40, 50 or 75, consecutive nucleotides of a sense strand of anucleic acid sequence according to the invention or of a correspondingantisense strand.

An “isolated” nucleic acid molecule is removed from other nucleic acidmolecules that are present in the natural source of the nucleic acid andcan moreover be essentially free from other cellular material or culturemedium, if it is being produced by recombinant techniques, or can befree from chemical precursors or other chemicals, if it is beingsynthesized chemically.

A nucleic acid molecule according to the invention can be isolated bymeans of standard techniques of molecular biology and the sequenceinformation supplied according to the invention. For example, cDNA canbe isolated from a suitable cDNA library, using one of the specificallydisclosed complete sequences or a segment thereof as hybridization probeand standard hybridization techniques (as described for example inSambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd edition, Cold Spring Harbor Laboratory, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). Inaddition, a nucleic acid molecule comprising one of the disclosedsequences or a segment thereof, can be isolated by the polymerase chainreaction, using the oligonucleotide primers that were constructed on thebasis of this sequence. The nucleic acid amplified in this way can becloned into a suitable vector and can be characterized by DNA sequenceanalysis. The oligonucleotides according to the invention can also beproduced by standard methods of synthesis, e.g. using an automatic DNAsynthesizer.

Nucleic acid sequences according to the invention, such as SEQ ID No: 7or derivatives thereof, homologs or parts of these sequences, can forexample be isolated by usual hybridization techniques or the PCRtechnique from other bacteria, e.g. via genomic or cDNA libraries. TheseDNA sequences hybridize in standard conditions with the sequencesaccording to the invention.

“Hybridize” means the ability of a polynucleotide or oligonucleotide tobind to an almost complementary sequence in standard conditions, whereasnonspecific binding does not occur between noncomplementary partners inthese conditions. For this, the sequences can be 90-100% complementary.The property of complementary sequences of being able to bindspecifically to one another is utilized for example in Northern blottingor Southern blotting or in primer binding in PCR or RT-PCR.

Short oligonucleotides of the conserved regions are used advantageouslyfor hybridization. However, it is also possible to use longer fragmentsof the nucleic acids according to the invention or the completesequences for the hybridization. These standard conditions varydepending on the nucleic acid used (oligonucleotide, longer fragment orcomplete sequence) or depending on which type of nucleic acid—DNA orRNA—is used for hybridization. For example, the melting temperatures forDNA:DNA hybrids are approx. 10° C. lower than those of DNA:RNA hybridsof the same length.

For example, depending on the particular nucleic acid, standardconditions mean temperatures between 42 and 58° C. in an aqueous buffersolution with a concentration of from 0.1 to 5×SSC (1×SSC=0.15 M NaCl,15 mM sodium citrate, pH 7.2) or additionally in the presence of 50%formamide, for example 42° C. in 5×SSC, 50% formamide. Advantageously,the hybridization conditions for DNA:DNA hybrids are 0.1×SSC andtemperatures of from about 20° C. to 45° C., preferably between about30° C. to 45° C. For DNA:RNA hybrids the hybridization conditions areadvantageously 0.1×SSC and temperatures of from about 30° C. to 55° C.,preferably from about 45° C. to 55° C. These stated temperatures forhybridization are examples of calculated melting temperature values fora nucleic acid with a length of approx. 100 nucleotides and a G+Ccontent of 50% in the absence of formamide. The experimental conditionsfor DNA hybridization are described in relevant genetics textbooks, forexample Sambrook et al., “Molecular Cloning”, Cold Spring HarborLaboratory, 1989, and can be calculated using formulas that are known bya person skilled in the art, for example depending on the length of thenucleic acids, the type of hybrids or the G+C content. A person skilledin the art can obtain further information on hybridization from thefollowing textbooks: Ausubel et al. (eds), 1985, Current Protocols inMolecular Biology, John Wiley & Sons, New York; Hames and Higgins (eds),1985, Nucleic Acids Hybridization: A Practical Approach, IRL Press atOxford University Press, Oxford; Brown (ed), 1991, Essential MolecularBiology: A Practical Approach, IRL Press at Oxford University Press,Oxford.

“Hybridization” can in particular be carried out under stringentconditions. Such hybridization conditions are for example described inSambrook, J., Fritsch, E. F., Maniatis, T., in: Molecular Cloning (ALaboratory Manual), 2nd edition, Cold Spring Harbor Laboratory Press,1989, pages 9.31-9.57 or in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6.

“Stringent” hybridization conditions mean in particular: incubation at42° C. overnight in a solution consisting of 50% formamide, 5×SSC (750mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6),5×Denhardt's solution, 10% dextran sulfate and 20 g/ml denatured,sheared salmon sperm DNA, followed by washing of the filters with0.1×SSC at 65° C.

The invention also relates to derivatives of the specifically disclosedor derivable nucleic acid sequences.

Thus, further nucleic acid sequences according to the invention can bederived e.g. from SEQ ID NO: 7 and can differ therefrom by addition,substitution, insertion or deletion of single or multiple nucleotides,but still code for polypeptides with the desired profile of properties.

The invention also encompasses nucleic acid sequences that comprise“silent” mutations or have been altered, in comparison with aspecifically stated sequence, according to the codon usage of a specialsource or host organism, as well as naturally occurring variants, e.g.splicing variants or allelic variants, thereof.

It also relates to sequences that can be obtained by conservativenucleotide substitutions (i.e. the amino acid in question is replaced byan amino acid of the same charge, size, polarity and/or solubility).

The invention also relates to the molecules derived from thespecifically disclosed nucleic acids by sequence polymorphisms. Thesegenetic polymorphisms can exist between individuals within a populationowing to natural variation. These natural variations usually produce avariance of 1 to 5% in the nucleotide sequence of a gene.

Derivatives of the nucleic acid sequence with the sequence SEQ ID NO: 7according to the invention mean for example allelic variants having atleast 60% homology at the level of the derived amino acids, preferablyat least 80% homology, very particularly preferably at least 90%homology, over the entire sequence range (regarding homology at theamino acid level, reference should be made to the details given abovefor the polypeptides). Advantageously, the homologies can be higher overpartial regions of the sequences.

Furthermore, derivatives are also to be understood to be homologs of thenucleic acid sequences according to the invention, in particular of SEQID NO: 7, for example fungal or bacterial homologs, truncated sequences,single-stranded DNA or RNA of the coding and noncoding DNA sequences.For example, homologs of SEQ ID NO: 7 have, at the DNA level, a homologyof at least 40%, preferably of at least 60%, particularly preferably ofat least 70%, very preferably of at least 80%, over the entire DNAregion given in SEQ ID NO: 7.

Moreover, derivatives are to be understood to be, for example, fusionswith promoters. The promoters that are upstream of the stated nucleotidesequences can be modified by at least one nucleotide exchange, at leastone insertion, inversion and/or deletion, though without, however,impairing the functionality or efficacy of the promoters. Moreover, theefficacy of the promoters can be increased by altering their sequence orthey can be replaced completely with more effective promoters, even oforganisms of a different genus.

3.3 Constructs According to the Invention

The invention also relates to expression constructs, comprising, underthe genetic control of regulatory nucleic acid sequences, a nucleic acidsequence coding for a polypeptide according to the invention; as well asvectors comprising at least one of these expression constructs.

“Expression unit” means, according to the invention, a nucleic acid withexpression activity, which comprises a promoter as defined herein and,after functional linkage to a nucleic acid that is to be expressed or agene, regulates expression, i.e. transcription and translation of thisnucleic acid or of this gene. In this context, therefore, it is alsocalled a “regulatory nucleic acid sequence”. In addition to thepromoter, other regulatory elements may be present, e.g. enhancers.

“Expression cassette” or “expression construct” means, according to theinvention, an expression unit which is functionally linked with thenucleic acid that is to be expressed or the gene that is to beexpressed. In contrast to an expression unit, an expression cassettethus comprises not only nucleic acid sequences which regulatetranscription and translation, but also the nucleic acid sequences whichshould be expressed as protein as a result of transcription andtranslation.

The terms “expression” or “overexpression” describe, in the context ofthe invention, production of or increase in intracellular activity ofone or more enzymes in a microorganism, which are encoded by thecorresponding DNA. For this, it is possible for example to insert a genein an organism, replace an existing gene with another gene, increase thenumber of copies of the gene or genes, use a strong promoter or use agene that codes for a corresponding enzyme with high activity, andoptionally these measures can be combined.

Preferably such constructs according to the invention comprise apromoter 5′ upstream from the respective coding sequence, and aterminator sequence 3′ downstream, and optionally further usualregulatory elements, in each case operatively linked to the codingsequence.

A “promoter”, a “nucleic acid with promoter activity” or a “promotersequence” mean, according to the invention, a nucleic acid which,functionally linked to a nucleic acid that is to be transcribed,regulates transcription of this nucleic acid.

“Functional” or “operative” linkage means, in this context, for examplethe sequential arrangement of one of the nucleic acids with promoteractivity and a nucleic acid sequence that is to be transcribed andoptionally further regulatory elements, for example nucleic acidsequences that ensure transcription of nucleic acids, and for example aterminator, in such a way that each of the regulatory elements canfulfill its function during transcription of the nucleic acid sequence.This does not necessarily require a direct linkage in the chemicalsense. Genetic control sequences, such as enhancer sequences, forexample, can also act on the target sequence from more remote positionsor even from other DNA molecules. Arrangements are preferred in whichthe nucleic acid sequence that is to be transcribed is positioneddownstream (i.e. at the 3′ end) of the promoter sequence, so that thetwo sequences are bound covalently to one another. The distance betweenthe promoter sequence and the nucleic acid sequence that is to beexpressed transgenically can be less than 200 base pairs, or less than100 base pairs or less than 50 base pairs.

Apart from promoters and terminators, examples of other regulatoryelements that may be mentioned are targeting sequences, enhancers,polyadenylation signals, selectable markers, amplification signals,origins of replication and the like. Suitable regulatory sequences aredescribed for example in Goeddel, Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990).

Nucleic acid constructs according to the invention comprise inparticular sequence SEQ ID NO: 7 or derivatives and homologs thereof, aswell as the nucleic acid sequences that can be derived from SEQ ID NO:8, which have advantageously been linked operatively or functionally toone or more regulatory signals for controlling, e.g. increasing, geneexpression.

In addition to these regulatory sequences, the natural regulation ofthese sequences can still be present upstream of the actual structuralgenes and optionally can have been altered genetically, so that naturalregulation has been switched off and expression of the genes has beenincreased. The nucleic acid construct can, however, also be of a simplerdesign, i.e. without any additional regulatory signals being insertedupstream of the coding sequence (e.g. SEQ ID NO: 7 or its homologs) andwithout removing the natural promoter with its regulation. Instead, thenatural regulatory sequence is mutated so that regulation no longertakes place and gene expression is increased.

A preferred nucleic acid construct advantageously also comprises one ormore of the aforementioned enhancer sequences, functionally linked tothe promoter, which permit increased expression of the nucleic acidsequence. Additional advantageous sequences, such as other regulatoryelements or terminators, can also be inserted at the 3′ end of the DNAsequences. One or more copies of the nucleic acids according to theinvention can be comprised in the construct. The construct can alsocomprise other markers, such as antibiotic resistances orauxotrophy-complementing genes, optionally for selection for theconstruct.

Examples of suitable regulatory sequences are comprised in promoterssuch as cos, tac, trp, tet, trp-tet, lpp, lac, lpp-lac, lacl^(q,) T7,T5, T3, gal, trc, ara, rhaP (rhaP_(BAD))SP6, lambda-P_(R) or in thelambda-P_(L) promoter, which are employed advantageously inGram-negative bacteria. Other advantageous regulatory sequences arecomprised for example in the Gram-positive promoters amy and SPO2, inthe yeast or fungal promoters ADC1, MFalpha, AC, P-60, CYC1, GAPDH, TEF,rp28, ADH. Artificial promoters can also be used for regulation.

For expression, the nucleic acid construct is inserted in a hostorganism advantageously into a vector, for example a plasmid or a phage,which permits optimum expression of the genes in the host. In additionto plasmids and phages, vectors are also to be understood as meaning allother vectors known to a person skilled in the art, i.e. for exampleviruses, such as SV40, CMV, baculovirus and adenovirus, transposons, ISelements, phasmids, cosmids, and linear or circular DNA. These vectorscan be replicated autonomously in the host organism or can be replicatedchromosomally. These vectors represent a further embodiment of theinvention.

Suitable plasmids are, for example in E. coli, pLG338, pACYC184, pBR322,pUC18, pUC19, pKC30, pRep4, pHS1, pKK223-3, pDHE19.2, pHS2, pPLc236,pMBL24, pLG200, pUR290, pIN-III¹¹³-B1, λgt11 or pBdCl; in StreptomycespIJ101, pIJ364, pIJ702 or pIJ361; in Bacillus pUB110, pC194 or pBD214;in Corynebacterium pSA77 or pAJ667; in fungi pALS1, pIL2 or pBB116; inyeasts 2alphaM, pAG-1, YEp6, YEp13 or pEMBLYe23 or in plants pLGV23,pGHlac⁺, pBIN19, pAK2004 or pDH51. The aforementioned plasmids representa small selection of the possible plasmids. Other plasmids are wellknown to a person skilled in the art and will be found for example inthe book Cloning Vectors (Eds. Pouwels P. H. et al. Elsevier,Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).

In a further embodiment of the vector, the vector comprising the nucleicacid construct according to the invention or the nucleic acid accordingto the invention can also be introduced advantageously in the form of alinear DNA to the microorganisms and integrated into the genome of thehost organism through heterologous or homologous recombination. Thislinear DNA can comprise a linearized vector such as a plasmid or justthe nucleic acid construct or the nucleic acid according to theinvention.

For optimum expression of heterologous genes in organisms, it isadvantageous to alter the nucleic acid sequences in accordance with thespecific codon usage employed in the organism. The codon usage caneasily be determined on the basis of computer evaluations of other,known genes of the organism in question.

The production of an expression cassette according to the invention isbased on fusion of a suitable promoter to a suitable coding nucleotidesequence and a terminator signal or polyadenylation signal. Commonrecombination and cloning techniques are used for this, as described forexample in T. Maniatis, E. F. Fritsch and J. Sambrook, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y. (1989) as well as in T. J. Silhavy, M. L. Berman and L. W.Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1984) and in Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Greene Publishing Assoc. and WileyInterscience (1987).

The recombinant nucleic acid construct or gene construct is insertedadvantageously in a host-specific vector for expression in a suitablehost organism, which enables the genes to be optimally expressed in thehost. Vectors are well known to a person skilled in the art and can befound for example in “Cloning Vectors” (Pouwels P. H. et al., Publ.Elsevier, Amsterdam-New York-Oxford, 1985).

3.4 Microorganisms that can be Used According to the Invention

Depending on the context, the term “microorganism” means the startingmicroorganism (wild-type) or a genetically modified, recombinantmicroorganism, or both.

By means of the vectors according to the invention, recombinantmicroorganisms can be produced, which have been transformed for examplewith at least one vector according to the invention and can be used forproduction of the polypeptides according to the invention.Advantageously, the recombinant constructs according to the invention,described above, are inserted in a suitable host system and expressed.Preferably, common cloning and transfection methods that are familiar toa person skilled in the art are used, for example co-precipitation,protoplast fusion, electroporation, retroviral transfection and thelike, in order to secure expression of the stated nucleic acids in theparticular expression system. Suitable systems are described for examplein Current Protocols in Molecular Biology, F. Ausubel et al., Publ.Wiley Interscience, New York 1997, or Sambrook et al. Molecular Cloning:A Laboratory Manual. 2nd edition, Cold Spring Harbor Laboratory, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

In principle, all prokaryotic or eukaryotic organisms can be consideredas recombinant host organisms for the nucleic acid according to theinvention or the nucleic acid construct. Microorganisms such asbacteria, fungi or yeasts are used advantageously as host organisms. Itis advantageous to use Gram-positive or Gram-negative bacteria,preferably bacteria of the families Enterobacteriaceae,Pseudomonadaceae, Rhizobiaceae, Streptomycetaceae or Nocardiaceae,especially preferably bacteria of the genera Escherichia, Pseudomonas,Streptomyces, Nocardia, Burkholderia, Salmonella, Agrobacterium orRhodococcus. The genus and species Escherichia coli is very particularlypreferred. Other advantageous bacteria can also be found in thefollowing group: alpha-proteobacteria, beta-proteobacteria orgamma-proteobacteria.

The host organism or host organisms according to the inventionpreferably comprise here at least one of the nucleic acid sequences,nucleic acid constructs or vectors described in this invention, whichcode for an enzyme with esterase activity as defined above.

The organisms used in the process according to the invention are grownor bred in a manner familiar to a person skilled in the art, dependingon the host organism. As a rule, microorganisms are grown in a liquidmedium, which comprises a source of carbon, generally in the form ofsugars, a source of nitrogen generally in the form of organic sources ofnitrogen such as yeast extract or salts such as ammonium sulfate, traceelements such as iron, manganese and magnesium salts and if appropriatevitamins, at temperatures between 0° C. and 100° C., preferably from 10°C. to 60° C., with oxygen aeration. The pH of the liquid nutrient mediumcan be maintained at a fixed value, i.e. regulated or not regulatedduring growing. Growing can be carried out batchwise, semi-batchwise orcontinuously. Nutrients can be supplied at the start of fermentation orcan be supplied subsequently, either semi-continuously or continuously.The ketone can be added directly during growing, or advantageously aftergrowing. The enzymes can be isolated from the organisms by the processdescribed in the examples or can be used as crude extract for thereaction.

3.5 Recombinant Production of the Esterase:

The invention also relates to processes for recombinant production ofpolypeptides according to the invention or functional, biologicallyactive fragments thereof, by cultivating a polypeptide-producingmicroorganism, if appropriate inducing expression of the polypeptidesand isolating them from the culture. The polypeptides can also beproduced on an industrial scale in this way, if so desired.

The microorganisms produced according to the invention can be culturedcontinuously or batchwise in a batch process or in a fed batch orrepeated fed batch process. A review of known culturing methods will befound in the textbook by Chmiel (Bioprocesstechnik 1. Einfuhrung in dieBioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in thetextbook by Storhas (Bioreaktoren and periphere Einrichtungen (ViewegVerlag, Braunschweig/Wiesbaden, 1994)).

The culture medium that is to be used must satisfy the requirements ofthe particular strains in an appropriate manner. Descriptions of culturemedia for various microorganisms are given in the handbook “Manual ofMethods for General Bacteriology” of the American Society forBacteriology (Washington D.C., USA, 1981).

These media that can be used according to the invention generallycomprise one or more sources of carbon, sources of nitrogen, inorganicsalts, vitamins and/or trace elements.

Preferred sources of carbon are sugars, such as mono-, di- orpolysaccharides. Very good sources of carbon are for example glucose,fructose, mannose, galactose, ribose, sorbose, ribulose, lactose,maltose, sucrose, raffinose, starch or cellulose. Sugars can also beadded to the media via complex compounds, such as molasses, or otherby-products from sugar refining. It may also be advantageous to addmixtures of various sources of carbon. Other possible sources of carbonare oils and fats such as soybean oil, sunflower oil, peanut oil andcoconut oil, fatty acids such as palmitic acid, stearic acid or linoleicacid, alcohols such as glycerol, methanol or ethanol and organic acidssuch as acetic acid or lactic acid.

Sources of nitrogen are usually organic or inorganic nitrogen compoundsor materials comprising these compounds. Examples of sources of nitrogeninclude ammonia gas or ammonium salts, such as ammonium sulfate,ammonium chloride, ammonium phosphate, ammonium carbonate or ammoniumnitrate, nitrates, urea, amino acids or complex sources of nitrogen,such as corn-steep liquor, soybean flour, soybean protein, yeastextract, meat extract and others. The sources of nitrogen can be usedseparately or as a mixture.

Inorganic salt compounds that may be present in the media comprise thechloride, phosphate or sulfate salts of calcium, magnesium, sodium,cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

Inorganic sulfur-containing compounds, for example sulfates, sulfites,dithionites, tetrathionates, thiosulfates, sulfides, but also organicsulfur compounds, such as mercaptans and thiols, can be used as sourcesof sulfur.

Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts can be used assources of phosphorus.

Chelating agents can be added to the medium, in order to keep the metalions in solution. Especially suitable chelating agents comprisedihydroxyphenols, such as catechol or protocatechuate, or organic acids,such as citric acid.

The fermentation media used according to the invention usually alsocomprise other growth factors, such as vitamins or growth promoters,which include for example biotin, riboflavin, thiamine, folic acid,nicotinic acid, pantothenate and pyridoxine. Growth factors and saltsoften come from complex components of the media, such as yeast extract,molasses, corn-steep liquor and the like. In addition, suitableprecursors can be added to the culture medium. The precise compositionof the compounds in the medium is strongly dependent on the particularexperiment and must be decided individually for each specific case.Information on media optimization can be found in the textbook “AppliedMicrobiol. Physiology, A Practical Approach” (Publ. P. M. Rhodes, P. F.Stanbury, IRL Press (1997) p. 53-73, ISBN 0 19 963577 3). Growth mediacan also be obtained from commercial suppliers, such as Standard 1(Merck) or BHI (Brain heart infusion, DIFCO) etc.

All media components are sterilized, either by heating (20 min at 1.5bar and 121° C.) or by sterile filtration. The components can besterilized either together, or if necessary separately. All thecomponents of the medium can be present at the start of growing, oroptionally can be added continuously or by batch feed.

The temperature of the culture is normally between 15° C. and 45° C.,preferably from 25° C. to 40° C. and can be kept constant or can bevaried during the experiment. The pH of the medium should be in therange from 5 to 8.5, preferably around 7.0. The pH for growing can becontrolled during growing by adding basic compounds such as sodiumhydroxide, potassium hydroxide, ammonia or ammonia water or acidcompounds such as phosphoric acid or sulfuric acid. Antifoams, e.g.fatty acid polyglycol esters, can be used for controlling foaming. Tomaintain the stability of plasmids, suitable substances with selectiveaction, e.g. antibiotics, can be added to the medium. Oxygen oroxygen-containing gas mixtures, e.g. ambient air, are fed into theculture in order to maintain aerobic conditions. The temperature of theculture is normally from 20° C. to 45° C. Culture is continued until amaximum of the desired product has formed. This is normally achievedwithin 10 hours to 160 hours.

The fermentation broth is then processed further. Depending on therequirements, the biomass can be removed completely or partially fromthe fermentation broth by separation techniques, e.g. centrifugation,filtration, decanting or a combination of these methods, or can be leftin the fermentation broth completely.

If the polypeptides are not secreted into the culture medium, the cellsmay also be disrupted and the product can be obtained from the lysate byknown techniques for isolating proteins. The cells can be disruptedoptionally by high-frequency ultrasound, by high pressure, e.g. in aFrench pressure cell, by osmolysis, by the action of detergents, lyticenzymes or organic solvents, by means of homogenizers or by acombination of several of the methods listed.

The polypeptides can be purified using known chromatographic methods,such as molecular sieve chromatography (gel filtration), Q-Sepharosechromatography, ion exchange chromatography and hydrophobicchromatography, and by other usual methods such as ultrafiltration,crystallization, salting-out, dialysis and native gel electrophoresis.Suitable methods are described for example in Cooper, F. G.,Biochemische Arbeitsmethoden, Verlag Walter de Gruyter, Berlin, N.Y. orin Scopes, R., Protein Purification, Springer Verlag, New York,Heidelberg, Berlin.

For isolating the recombinant protein it may be advantageous to usevector systems or oligonucleotides, which extend the cDNA by definednucleotide sequences and therefore code for modified polypeptides orfusion proteins, which can be used e.g. for simpler purification.Suitable modifications of this kind are for example so-called “tags”which function as anchors, e.g. the modification known as thehexa-histidine anchor, or epitopes that can be recognized as antigens byantibodies (described for example in Harlow, E. and Lane, D., 1988,Antibodies: A Laboratory Manual. Cold Spring Harbor (N.Y.) Press). Theseanchors can provide adhesion of the proteins to a solid support, e.g. apolymer matrix, for example for packing a chromatographic column, or canbe used on a microtiter plate or on some other support.

At the same time, these anchors can also be used for recognition of theproteins. For recognition of the proteins it is also possible to useordinary markers, such as fluorescent dyes and enzyme markers which forma detectable reaction product after reaction with a substrate, orradioactive markers, alone or in combination with the anchors forderivatization of the proteins.

3.6 Applications According to the Invention of the Esterases

The invention also relates to processes for enantioselective esterhydrolysis using the esterase, which processes comprise contacting theesterase with a stereoisomer mixture of an optically active ester of theformula I and obtaining the optically active compounds produced bystereoselective hydrolysis of any of the two stereoisomers and/or thenonhydrolyzed ester enantiomer from the reaction medium. It is, however,also possible for the esterase to hydrolyze those esters of the formulaI which are not optically active.

The invention also relates to processes for enantioselectivetransesterification, which comprises contacting a stereoisomer mixtureof an optically active alcohol of the formula II with an ester of theformula I in the presence of the esterase, and obtaining the unreactedalcohol stereoisomer from the reaction medium, or contacting astereoisomer mixture of an optically active ester of the formula I withan alcohol of the formula II in the presence of said esterase, andobtaining a stereoisomer of the optically active alcohol comprised insaid ester from the reaction medium. Vinyl esters are preferably used intransesterification as acylating agents for an optically active alcohol.This is advantageous because, after the conversion, the alcohol functionof the vinyl ester is no longer available for the reverse reaction dueto tautomerization. The esterase also catalyzes transesterificationprocesses in which neither the ester nor the alcohol is opticallyactive.

Preferred substrates of ester hydrolysis are esters of ethanol,propanol, butanol and, particularly preferably, butynol esters (butynolesters, esters of 1-methylprop-2-ynol) with carboxylic acids such as,for example, acetic acid, propionic acid, butyric acid, pentanoic acid,hexanoic acid, heptanoic acid, octanoic acid, lactic acid,2-ethylhexanoic acid, 3-methylbutyric acid, methoxyacetic acid,2-methylpropionic acid, 2-butenoic acid, 3-chloropropionic acid and2-methylpentanoic acid. Particular preference is given to butynylbutyrate and butynyl methylbutyrate.

Preferred alcohols in the transesterification are ethanol, propanol andbutanol, particularly preferred is butynol.

Preferred esters in the transesterification are vinyl esters such as,for example, vinyl acetate, vinyl propionate and vinyl butyrate.

Reaction media used in the above methods are organic solvents such as,for example, alkanes, ethers, toluene, dioxane, methyl isobutyl ketone,methyl tert-butyl ether (MTBE) and the like. In the ester hydrolysis,mixtures made from the buffer solution used and organic solvents suchas, for example, MTBE and heptane or toluene may also be used.

Racemate resolution, i.e. enantioselectivity, and reaction rate can beinfluenced via size and hydrophobicity of the acid moiety.

The reaction according to the invention is preferably carried out atroom temperature at from pH 6 to 9, particularly preferably at from pH7.0 to 7.4. The esterase may be employed in the form of isolated orpurified enzyme, as cells of the microorganism expressing the esterase,as culture supernatant, cell lysate or extract of said microorganism, oras immobilized esterase. The reaction products can be isolated from thereaction solution by chemical or physical separation processes known tothe skilled worker. The esterase can be isolated from the reactionmixture by membrane filtration.

It is possible to immobilize the esterase with the aid ofpolyacrylamide, alginic acid or carrageenans. It is also possible tobind the esterase covalently or by adsorption to suitable carriers bymeans of known methods. The esterase is preferably immobilized bylyophilization on kieselguhr or by ammonium sulfate precipitation.

EXPERIMENTAL SECTION

Unless stated otherwise, the cloning steps carried out within the scopeof the present invention, for example restriction cleavages, agarose gelelectrophoresis, purification of DNA fragments, transfer of nucleicacids to nitrocellulose and nylon membranes, linking of DNA fragments,transformation of microorganisms, culturing of microorganisms,propagation of phages and sequence analysis of recombinant DNA, may becarried out as described in Sambrook et al. (1989), loc. cit.

Example 1 Selection of an Esterase-Expressing Pseudomonas glumae Mutant

Starting point of the screening was the lipase-producing strainPseudomonas (Burkholderia) glumae LU8093. The lipase produced by saidstrain makes it possible to carry out a number of interesting reactions(Balkenhohl, F. et al., J. prakt. Chem. 339, (1997), 381-384). Lacticesters, methylbutyric esters and phenylacetic esters, however, are notsubstrates for the lipase and cannot be hydrolyzed by said strain in anyother way either.

The hydrolysis products are, however, usable as carbon source.Therefore, mutants of LU8093 were sought which are able to hydrolyzesaid esters and to grow using the hydrolysis products as carbon source.Mutants with novel esterase activity should therefore reveal themselvesby growing on said esters.

Selection Conditions:

LU8093 was cultured on medium for 16 h and harvested by centrifugation.The cells were washed twice with saline. 10⁶ cells were plated out ontominimal medium plates comprising 0.5 or 1.0 g/l ethyl phenylacetate asthe sole carbon source. Initially, however, there was no growth. Onlyafter 4 to 6 days were single colonies recognizable. Their numberincreased further over the following days.

From the esterase-positive mutants obtained in this way, the mutantLU2023 was selected. Surprisingly, the novel esterase activity was alsosuitable for selective hydrolysis of small organic molecules. As anexample, selective hydrolysis was shown for butynol ester.

Example 2 Fermentation of Pseudomonas glumae LU2023

To obtain the esterase, Pseudomonas glumae LU2023 was cultured on a 14-1scale and the active biomass was harvested.

In the laboratory, Pseudomonas glumae LU2023 was streaked onto agarplates with M12 mineral salt medium and 1 g/l EPA and incubated at 28°C. for 36 to 48 hours. The plates were then stored at 4° C. for fourweeks.

Fermentation of the strain was carried out in an Infors xxy 14 lfermenter. For the preculture, 250 ml of medium were inoculated with 2to 3 Pt loops and incubated at 200 rpm and 28° C. for 24 hours. The mainculture was carried out under the following conditions:

Temperature 28° C.

Air feed 7 l/min

Stirring 600 rpm

Fermentation run time about 24 h

Built-in pH and pO₂ measurements

Medium for Preculture and Main Culture

15 g/l Springer yeast autolysate 65%

1.6 g/1 magnesium sulfate×7 water

0.02 g/l calcium chloride×2 water

3.5 g/l potassium dihydrogen phosphate

3.5 g/l dipotassium hydrogen phosphate

5 g/l diammonium hydrogen phosphate

6 ml Pluriol P2000 antifoam

The above ingredients were dissolved in deionized water and the solutionwas adjusted to pH 6.5 using 25% strength ammonia solution. 5 ml/l traceelement solution and 2 g/l glucose were sterile-filtered separately.

After sterilizing and completing the medium, 0.5 g/l ethyl phenylacetatewas introduced into the fermenter. Addition of Pluriol P2000 controlledthe foam appearing during fermentation. Fermentation was stopped whenthe pO₂ in the fermenter increased again to above 85%. The fermentercontents were then centrifuged at below 15° C. and about 9000 to 10 000g, and the clear effluent was discarded. The cell mass was frozen at−16° C.

Example 3 Purification of the Esterase from Pseudomonas glumae LU2023

Pseudomonas glumae (LU2023) cells (100 ml, wet weight: 50 g) were lysedin a glass bead mill (100 ml of glass beads, diameter: 0.5 mm) at 4° C.and 3000 rpm. After centrifugation (10 000 rpm, 30 min) and washing theglass beads, the supernatant (300 ml) was subjected to manganesechloride precipitation (pH 7 to 7.5; final concentration: 50 mM). Afteranother centrifugation, the supernatant was adjusted to pH 8.0 and EDTAwas added at a concentration of 50 mM. This volume was purified byQ-Sepharose (300 ml) chromatography. After applying the sample, thecolumn was washed with 50 mM Tris/HCl. The fraction of interest wascollected and concentrated by ultrafiltration (100 kDa). Thebutynol-hydrolyzing esterase was separated from a nonspecific esteraseby molecular sieve chromatography (diameter: 5 cm, height: 90 cm;material: S-300). The active fraction obtained was cloudy and was againconcentrated. The esterase was obviously membrane-bound. The membranefraction was then first digested by a protease (trypsin, weight ratio:1:50 to 1:100). This caused all proteins to disappear from the membranefraction apart from a few bands in the SDS polyacrylamide gelelectrophoresis. The activity was preserved. Said bands were separatedfrom one another by native gel electrophoresis (0.04% SDS), and anactivity assay identified the esterase in this native gel. Said esterasewas eluted from the gel and then appeared as a clean band in adenaturing SDS polyacrylamide gel electrophoresis.

The protein purified in this way was transferred by blotting onto a PVDFmembrane and sequenced, or, after trypsin cleavage, the peptides wereseparated by reversed phase HPLC and sequenced. Since the amino terminusof the protein was blocked, only tryptic peptides were obtained. Thevarious amino acid sequences showed weak homologies to a muconatecycloisomerase, EC 5.5.1.1, from Acinetobacter lwoffii and Pseudomonasputida, and also to lactone esterase from Pseudomonas fluorescens. Thepeptide having the sequence AIDAIFAPEGV (SEQ ID NO: 24) showed homologyto pectin esterases (EC 3.1.1.11).

The drawing in FIG. 1 depicts a sequence alignment of a partial aminoacid sequence according to the invention sequence to a partial sequenceof a lactone-specific esterase from Pseudomonas fluorescens.

Example 4 Immobilization of Esterase

Various methods were employed for the immobilization.

-   1. The esterase was substantially inactivated by precipitation with    acetone in the presence of kieselguhr. 25 mg of protein were mixed    with 3.5 g of kieselguhr (Merck), and 1.4 l of acetone (−20° C.)    were added for 10 minutes. The loaded support was then removed via a    G3 glass suction filter, the filter residue was washed with cold    acetone and dried.-   2. The esterase does not bind to Accurel (Akzo).-   3. It was possible to immobilize the esterase (2.3 units/g, EPA    assay) on kieselguhr by lyophilization. For this, the enzyme    solution was mixed with kieselguhr and frozen at −80° C.    Subsequently, the solid substance was dried by lyophilization.-   4. The esterase (454 milliunits/g, EPA assay) was immobilized by    ammonium sulfate precipitation. For this, the enzyme was    precipitated at 80% saturation of ammonium sulfate in the presence    of kieselguhr.

Example 5 Racemate Resolution using the Esterase from Pseudomonas glumaeLU2023

Procedure (Standard Approach)

100 units of esterase were reacted with 20 mmol of butynol butyrate(1-methylprop-2-ynyl butyrate) in phosphate buffer (200 ml, 10 mM, pH7.4) with stirring. The pH was continuously measured and kept at approx.pH 7.4 by adding sodium hydroxide solution. At the times indicated intable 1, samples were taken and extracted twice with methyl tert-butylether (MTBE), and the organic phase was analyzed by GC (Chiraldex GTA).The esterase could be removed from the reaction mixture by membranefiltration.

With its concentration increasing, the less preferred ester enantiomerwas increasingly converted. After about 45 minutes, this caused a dropin the ee of S-butynol in the reaction mixture. The ee of the productreached its maximum at 84% (83-97.9%) after approx. 30 to 40 minutes.The ee of the substrate increased to over 99% over the course of 90minutes. The ee (enantiomer excess) is defined as the amount of thepreferably converted enantiomer in percent minus the amount of the lesspreferably converted enantiomer in percent. In most cases, thiscorresponds to the optical purity. The drop in pH was linear up to 30minutes. From approx. 100 minutes onward, the pH change was negligible.

After the extraction, the residual esterase activity in the aqueousphase was still approx. 50%.

TABLE 1 ee of substrate Ester ee of product (R)- conversion Time(S)-butynol butynol ester in % (corr.)  0 min Nd 5.20 nd  7 min Nd 10.20nd 13 min 75.50 20.40 12 20 min 81.80 29.10 16 26 min 83.90 42.00 22 32min 84.60 53.70 27 45 min 84.00 78.80 36 70 min 70.80 97.10 47 90 min69.60 99.10 52 121 min  63.10 99.40 56 150 min  52.00 99.50 67

Table 1 shows the time-dependent enantiomer excess on conversion ofbutynol butyrate by the esterase. According to the R/S convention byCahn, Prelog and Ingold, R and S configurations define the twoenantiomers of a chiral molecule. The conversion is the proportion ofconverted ester in the reaction mixture.

Example 6 Dependence of the Esterase Specificity on Size andHydrophobicity/Charge of the Acid Moiety of the Ester

Standard Approach

100 units of esterase were reacted with 20 mmol of butynol ester inphosphate buffer (200 ml, 10 mM, pH 7.4) with stirring. The pH wascontinuously measured and kept at pH 7.0 by continuous titration.Samples taken were extracted twice with methyl tert-butyl ether (MTBE),and the organic phase was analyzed by GC (Chiraldex GTA).

Result

The quality of racemate resolution and the reaction rate depended onsize and hydrophobicity of the acid moiety. The best substrates forbutynol esterase were butynol butyrate and butynol methylbutyrate.Lipases are inactive with these substrates. This is also true forlong-chain esters such as butynyl n-decanoate.

TABLE 2 Acid component ee [%] Conversion [%] E Acetate 73 (S) 48 12Butyrate 95 (S) 36 67 Pentenoate 74 (S) 47 13 Hexanoate 66 (S) 44 8Octanoate 64 (S) 43 8 2-Ethylhexanoate no conversion Phenylacetate 51(S) 12 3 3-Phenylpropionate 73 (S) 44 11 3-Cyclohexylpropionate 22 (S)18 2

Table 2 shows the dependence of the enantiomer excess for the conversionof esters by the esterase on the acid moiety of the converted ester.

Example 7 Transesterification in Organic Medium using the Esterase

10 mmol of rac-butynol and 5 mmol of vinyl butyrate were dissolved in 50ml of methyl tert-butyl ether (MTBE) and mixed with 9 units of esterase(3.3 g) supported on kieselguhr, and the mixture was shaken at roomtemperature for 24 h. After filtration, the solvent was removed and theproduct mixture was characterized by GC.

At 47% conversion, (R)-butynol (18% ee) and the butyrate of (S)-butynol(45% ee) remained.

In methyl isobutyl ketone, (R)-butynol with 16% ee and the butyrate of(S)-butynol with 52% ee were obtained at 43% conversion.

Table 3 shows the dependence of the enantiomer excess for the conversionof esters by the esterase on the acid moiety of the converted ester.

TABLE 3 Mixture Temp Buffer system No. Substrate pH [° C.] sol. [mmol/l]Additives ee¹⁾ 8 Butynyl n-decanoate 7.0 RT Phosphate 10 none 54.37 14Butynyl n-pentanoate 7.0 RT Phosphate 10 none 80.40 15 Butynyl2-ethylhexanoate 7.0 RT Phosphate 10 none 81.77 16 Butynyl butyrate 7.0RT Phosphate 10 none 83.90 17 Butynyl butyrate 7.0 RT Phosphate 10 0.5%Triton 80.83 18 Butynyl n-hexanoate 7.0 RT Phosphate 10 0.5% Triton78.63 19 Butynyl n-octanoate 7.0 RT Phosphate 10 0.5% Triton 74.70 20Butynyl butyrate 7.0 RT Phosphate 10 10% n-Propanol 87.47 21 Butynylbutyrate 7.0 RT Phosphate 10 1M NaCl 85.70 23 Butynyl n-pentanoate 7.0RT Phosphate 10 0.5% Triton 84.40 24 Butynyl butyrate 6.0 RT Phosphate10 none 85.37 25 Butynyl butyrate 8.0 RT Tris 10 none 85.33 26 Butynylbutyrate 7.0 10 Phosphate 10 none 85.90 27 Butynyl butyrate 7.0 37Phosphate 10 none 75.67 28 Butynyl 3-methylbutyrate 7.0 RT Phosphate 10none 90.50 29 Butynyl methoxyacetate 7.0 RT Phosphate 10 none 76.33 31Butynyl butyrate 7.0 RT Phosphate 10 none 85.00 32 Butynyl butyrate 7.0RT Phosphate 10 2-phase system 84.93 33 Butynyl 3-methylbutyrate 7.0 RTPhosphate 10 2-phase system 92.70 34 Butynyl 2-methylpropionate 7.0 RTPhosphate 10 none 89.17 35 Butynyl 2-butenoate 7.0 RT Phosphate 10 none76.03 36 Butynyl 3-chloropropionate 7.0 RT Phosphate 10 none 71.13 40Butynyl 2-methylpentanoate 7.0 RT Phosphate 10 none 85.93 ¹⁾Averages ofthe 3 best values for ee of S-butynol

Example 8 Preparation of a Gene Library of Strain LU2023 and Cloning ofLU2898

a) Obtaining Chromosomal DNA:

LU2023 (cf. examples 1 and 2 above) was grown in 100 ml of FP medium(Becton Dickinson GmbH) at 28° C. and 180 rpm for 24 h. The cells wereharvested by means of centrifugation (2000 rpm/5 min), resuspended in 5ml of lysis buffer 1 (0.41 M sucrose, 0.01 M MgSO₄*7H₂O, 50 ml/L M12medium (10× conc.), 10 mL 10% KH₂PO₄ pH 6.7, 2.5 mg/ml lysosyme [addshortly before use]) and incubated at 37° C. for approx. 4 h. Aftercentrifugation (2000 rpm/20 min), the resulting protoplasts are washedin 5 ml of lysis buffer 1 without lysosyme (2000 rpm/20 min) andresuspended in 10 mM TRIS-HCl pH 8.0.

After another washing with 10 mM TRIS-HCl pH 8.0 (3000 rpm/5 min), thepellet was resuspended in 4 ml of TE buffer (10 mM TRIS-HCl, 1 mM EDTA,pH 8) and mixed with 0.5 ml each of SDS (10% w/v) and NaCl (5M). Thiswas followed by adding 100 μl of proteinase K solution (200 μg/ml) andincubation at 37° C. for 16 hours. Subsequently, TE buffer was added tothe solution to a final volume of 10 ml. This solution was admixed 1:1with phenol. After centrifugation (4000 rpm/5 min), the upper phase wasremoved and mixed with a mixture of phenol, chloroform and isoamylalcohol (12:12:1). The upper phase was removed and mixed with one volumeof chloroform isoamyl alcohol (24:1). This procedure was repeated untilthe upper phase was clear.

DNA was precipitated from the upper phase by adding 2 volumes of ethanoland 1/50 volumes of CH₃COONa (3M) at −20° C. (duration: approx. 30 min)and pelleted by centrifugation (12 000 rpm, 30 min, 4° C.) andresuspended in TE buffer. RNase (1 ml of a 20 μg/ml solution) hydrolyzesRNA at 37° C. within 1 h. The solution is then dialyzed against TEbuffer at 4° C. three times for 1 hour each. The DNA is precipitated inaliquots of 0.4 ml each by adding ethanol (2 volumes plus ⅓ volume ofLiCl, 2M) and incubation at −20° C. for 30 minutes. Centrifugation (15000 rpm, 30 min, 4° C.) pelleted the DNA which was then dried. The DNAof a 0.4 ml aliquot was resuspended in 0.5 ml TE buffer.

b) Restriction of DNA and Cloning

1 μg of chromosomal DNA was digested with 120 U PstI at 37° C. for 180min. 0.4 μg of pUC19 (e.g. New England Biolabs) was restricted with 160U of PstI (1 h at 37° C.) and subsequently dephosphorylated withalkaline phosphatase (1 h at 37° C. plus inactivation at 65° C. for 15minutes). The DNA fragments were ligated (2 h at room temperature) usingthe Rapid Ligation Kit (Roche). The ligation mixtures were transformedinto transformation-competent Escherichia coli (Stratagene) according tothe manufacturer's information. The cells were streaked out ontoIPTG/X-Gal FP plates (FP medium containing 100 μg/ml ampicillin) andincubated at 37° C. for 16 hours. White colonies were streaked out ontofresh FP plates containing ampicillin.

c) Gene Library Screening

The colonies were transferred to Whatman filter paper using a sterilevelvet cushion. The filters were placed in 2.5 ml of assay solution (10mM TRIS*HCl, pH 7.5, 0.01% bromocresol purple, 0.1% racemic ester[rac-methyl lactate (MEE) or rac-ethyl methylbutyrate or rac-ethylphenylacetate]). Colonies whose cells have esterase activity changecolor from blue to yellow within 5 minutes. Among the 2900 coloniesassayed there was one which caused a strong color change. This strainwas referred to as LU2898. FIG. 2 depicts a diagrammatic representationof the cloning strategy of LU2898.

Example 9 Sequence Analysis of Recombinant DNA of LU2898

The plasmid from LU2898 has a 7.7 kb insertion. The plasmid harboringthe butynol esterase gene E. coli LU2898 was completely sequenced. Afirst search in DNA databases produced an open reading frame (positions1394 to 2923), with the insert having a homology to hydrolytic enzymes.

The ERGO database (Integrated Genomics, Inc. ©1999-2004, Chicago, USA)was searched with the DNA sequence of LU2898 butynol esterase. Only theregion from 1384 to 2397 by of the butynol esterase gene was found to behomologous to known hydrolases.

The region from 1384 to 2397 by is obviously the DNA of the desiredbutynol esterase. Another good indication is the GXSXG motif which istypical for serinecarboxyl esterases and which is located in amino acidpositions 127-131 in the LU2023 butynol esterase.

The good agreement between the LU2898 butynol esterase gene and the B.cepacia hydrolase ends from about position 2400. Further down thereading frame, there is a very distinct homology to the 3′ ends of NADPreductases.

These results are illustrated by attached FIG. 3. FIG. 3 is a summary ofthe ERGO analyses. The abrupt transition of homologies in the LU2898butynol esterase coincides with a PstI cleavage site. Cleavage sites forthe PstI restriction enzyme were not expected for the cloning strategychosen. This enzyme was used for completely cleaving LU2023 chromosomalDNA for preparing the plasmid library. After complete hydrolysis,internal recognition sites for PstI should no longer be present. Thefinding of PstI cleavage sites in the LU2898 plasmid can be explained bythe fact that different gene fragments produced by said PstI restrictionwere connected to one another during plasmid construction and eventuallyligated into the PstI cleavage site of pUC19, meaning that the readingframe assigned to butynol esterase possibly represents only part of theactual gene from Ps. glumae LU2023. The butynol esterase gene has beendisrupted at the PstI site and subsequently connected to a fragment ofan NADP reductase gene.

This hypothesis is supported by protein sequencing data. Ps. glumaeLU2023 butynol esterase has been purified. The amino acid sequence ofthe protein was determined by Edman degradation. The complete sequenceis known except a few N- and C-terminal amino acids. It corresponds tothe amino acid sequence from the first part of butynol esterase, derivedfrom the DNA data of LU2898. The experimentally determined molecularweight of butynol esterase of 41.3 kDa is markedly lower than the valuederived from the DNA sequence of the recombinant LU2898 butynol esterasegene. According to the DNA sequence data, butynol esterase should have amolecular weight of 55 kDa.

Evidence was found with the aid of the database analysis that thebutynol esterase has not been cloned in its full length. The plasmidfrom LU2898 does not comprise the 3′ end of the butynol esterase gene.

Example 10 Finding of Clone LU11147 Comprising a Truncated 335AA ButynolEsterase Mutant

The sequence data of the plasmid from LU2898 were utilized in order todesign the following PCR primers.

Breu0811:       HindIII GGCGAGAAGCTTAGAAATCATGATCGTCCA (SEQ ID NO: 20)Breu0812:       XbaI GGATCCTCTAGAGTCTCACTGCAGCGGGCC (SEQ ID NO: 21)

These oligonucleotides were used to amplify by means of PCR the DNA fromLU2898, which has the above-described homology to esterase.

PCR Conditions:

5 μl of 10*Taq polymerase buffer 125 ng of primer Breu0811 125 ng ofprimer Breu0812 2 μl of dNTP (10 mM) 50 ng of plasmid DNA from LU111472.5 μl of DMSO ad 50 μ of H₂O hot start of Taq-DNA polymerase (RocheDiagnostics) with: 0.5 UPCR Temperature Program:

 5 min, 95° C., 45 s, 55° C.,  3 min, 72° C., {close oversize brace} (30cycles) 30 s, 95° C., 45 s, 55° C., 10 min, 72° C., ∞, 4° C.

After restriction with HindIII and XbaI, the PCR product is ligated intoan appropriately prepared pUC19 (e.g. New England Biolabs).

The plasmid vector prepared in this way was then used for transformingE. coli XL1 blue. The ligation mixtures were transformed intotransformation-competent Escherichia coli (Stratagene) according to themanufacturer's information. The cells were streaked out onto IPTG/X-GalFP plates (FP medium containing 100 μg/ml ampicillin) and incubated at37° C. for 16 hours. Plasmid DNA was prepared from the transformants(Birnboim et al., Nucleic Acids Research, 1979, 7, 1513) and checked forsuccessful insertion of the PCR product by means of control restrictionwith HindIII and XbaI.

Example 11 Expression of the Truncated 335 AA Butynol Esterase

The esterase gene was subsequently cloned into the vector pDHE1650(described in WO-A-2004/050877), using the NdeI and HindIII cleavagesites.

To this end, the esterase gene was first amplified using the followingforward and reverse primers:

Breu1499 TATACATATGATCGTCCAACTGATCGCCATCGTG, Breu1500ATTTAAGCTTTTACTGCAGCGCGCCGGCCTGCGTGACCTC

followed by restriction digest using NdeI and HindIII. The insert wasligated into the pDHE1650 vector which previously had been predigestedwith the same restriction enzymes (NdeI and HindIII). In the same way,the place holder gene present in pDHE1650 was replaced with the esterasegene of the invention. Two transformants were isolated and the plasmidswere extracted by DNA precipitation in a manner known per se. Saidplasmids were referred to as pDHE1650-Est2 and pDHE1615-Est4.

For protein expression, the plasmid carrying the esterase gene wastransformed into Escherichia coli TG1 (DSMZ No. 6056), using the TSSmethod ([Chung C T, Niemela S L and Miller R H, 1989. One-steppreparation of competent Escherichia coli: Transformation and Storage ofBacterial Cells in the Same Solution. Proc. Natl. Acad. Sci. U.S.A. 86:2172-2175). A single colony was isolated and propagated in 5 ml ofLB/Amp/Tet/Spec/Cm medium (=LB medium according to Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular cloning: A Laboratory Manual.2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989 plus 100 μg/ml ampicillin, 10μg/ml tetracycline, 100 μg/ml spectinomycin and 20 μg/mlchloramphenicol) at 37° C. and 250 rpm overnight. 100 ml ofLB/Amp/Tet/Spec/Cm medium supplemented with 10 ml/l trace elementsolution were inoculated with 500 μl of the overnight culture andpropagated at 37° C. and 200 rpm. (The trace element solution iscomposed as follows: FeSO₄*H₂O 200 mg/l, ZnSO₄*7H₂O 10 mg/l, MnCl₂*4H₂O3 mg/l, H₃BO₃ 30 mg/l, CoCl₂*6H₂O 20 mg/l, CuCl₂*6H₂O 1 mg/l, NiCl₂*6H₂O 2 mg/l, Na₂MoO₄*2H₂O 3 mg/l, Titriplex III 500 mg/l). Proteinexpression was induced with 1 mM L-Rhamnose once OD₆₀₀ had reached avalue of from 0.5 to 0.6. The temperature was reduced to 30° C., whilethe rate of rotation was kept at 200 rpm. The culture was culturedovernight. Subsequently, the cells were centrifuged (15 min, 4° C., 3220g) and the cell pellet was stored at −20° C. overnight.

The cells were disrupted by resuspending the cell pellet (of 50 ml of TBculture) in sodium phosphate buffer (25 mM, ionic strength 154 mM, pH7.5). The cells were disrupted by homogenization (1500 bar, 2 passages).Cell fragments were removed by centrifugation (30 min, 4° C., 20 817 g).The supernatant was applied to a Protein 200 Plus Labchip and analyzedfor protein using the Agilent 2100 Bioanalyzer. The removed cellfragments were resuspended in 8 M urea, followed by centrifugation (30min, 4° C., 20 817 g) in order to remove undissolved particles. Theclear supernatant (membrane fraction) was applied to a Protein 200Plus-Labchip and analyzed for protein using the Agilent 2100Bioanalyzer.

Example 12 Preparation of Butynol Esterase (335AA) Mutants

a) Materials

All chemicals used were suitable for analytical purposes or had a higherquality and were purchased from Sigma-Aldrich Chemie, Taufkirchen,Germany, Applichem (Darmstadt, Germany) and Carl Roth (Karlsruhe,Germany).

A thermocycle (Mastercycler gradient, Eppendorf, Hamburg, Germany) andthin-wall PCR tubes (Multi-Ultra tubes; 0.2 ml; Carl Roth) were used inall PCR experiments. The PCR volume was in each case 50 μl; largervolumes were prepared by way of multiple 50 μl PCR mixtures. The amountof DNA used was quantified using a NanoDrop photometer (NanoDropTechnologies, Wilmington, Del.).

b) Cloning of Butynol Esterase Gene into pASK-IBA7 Vector:

The pASK-IBA7 vector (IBA GmbH, Göttingen, Germany) (SEQ ID NO: 19,plasmid for protein expression with N-terminal Strep-tag) was amplifiedusing the following primers:

Forward primer (SEQ ID NO: 9) 5′-TTTTTGCCCTCGTTATCTAGATTT-3′ Reverseprimer (SEQ ID NO: 10) 5′-CCGGAATTCCGGTATCTAACTAAGCTTGACCTG-3′

The 50 μl PCR mixture (95° C. 3 min; 1 cycle//95° C. 30 s, 56.2° C. 45s, 72° C. 7 min; 30 cycles//72° C. 10 min; 1 cycle) comprised: 20 μmolof forward primer, 20 μmol of reverse primer, 0.2 mM of each dNTP (RocheDiagnostics GmbH, Mannheim, Germany), 150 ng of pASK-IBA7 plasmid and2.5 U of Pfu DNA polymerase (Fermentas GmbH, St. Leon-Rot, Germany). Theproduct was purified by gel, using the QIAquick gel extraction kit(Qiagen, Hilden, Germany) and again amplified under conditions identicalto those above. After gel purification (QIAquick gel extraction kit,Qiagen), 2 μg of the DNA product were digested with 40 U of EcoRI (NewEngland Biolabs GmbH, Frankfurt, Germany) in a 100 μl reaction mixtureat 37° C. for 4 hours. The digested product was PCR-purified using theQIAquick PCR purification kit (Qiagen).

The butynol esterase gene was amplified using the following primers:

Forward primer (SEQ ID NO: 11) 5′-[Phos]GACCATGATTACGCCAAGCTTGC-3′Reverse primer (SEQ ID NO: 12) 5′-CCGGAATTCCGGTCACTGCAGCGCGCCGGCCTG-3′.

The 50 μl PCR mixture (95° C. 2 min; 1 cycle//95° C. 30 s, 59° C. 45 s,72° C. 3 min; 30 cycles//172° C. 10 min; 1 cycle) comprised: 20 μmol offorward primer, 20 μmol of reverse primer, 0.2 mM of each dNTP (RocheDiagnostics GmbH); 150 ng of plasmid LU11147 (pUC19 vector, carrying thebutynol esterase gene), 0.02% DMSO and 2.5 U of Pfu DNA polymerase(Fermentas). After gel purification (QIAquick gel extraction kit,Qiagen), 2 μg of the DNA product were digested with 40 U of EcoRI (NewEngland Biolabs GmbH, Frankfurt, Germany) in a 100 μl reaction mixtureat 37° C. for 4 hours.

The digested product was PCR-purified using the QIAquik PCR purificationkit (Qiagen).

The butynol esterase gene was ligated into the vector using blunt andEcoRI cleavage sites. The reaction mixture (20 μl) comprised: 20 ng ofvector, 120 ng of insert and 2 U of T4 DNA ligase (Roche DiagnosticsGmbH). The reaction mixture was first incubated at room temperature for1 hour, followed by overnight incubation in a refrigerator. Theresulting plasmid was referred to as pASK-IBA7 esterase.

c) Preparation of the First Butynol Esterase Mutant Library:

The butynol esterase gene was first amplified by means of error-pronePCR (95° C. 2 min; 1 cycle//95° C. 30 s, 59° C. 45 s, 72° C. 3 min; 40cycles//72° C. 10 min). The following primers were used:

Forward primer: 5′-CGACAAAAATCTAGATAACGAGGGCAA-3′ (SEQ ID NO: 13)Reverse primer: 5′-TTGACTTCACAGGTCAAGCTTAGTTAG-3′. (SEQ ID NO: 14)

The reaction mixture (50 μl) comprised: 20 μmol of forward primer, 20μmol of reverse primer, 0.2 mM of each dNTP (Roche Diagnostics GmbH),100 ng of plasmid pASK-IBA7 esterase, 0.02% DMSO, 0.1 mM MnCl₂ and 2.5 Uof Tag DNA polymerase (Qiagen). After gel purification (QIAquick GelExtraction Kit; Qiagen), 4 μg of DNA were double-digested with 40 U ofEcoRI (New England Biolabs GmbH) and 30 U of XbaI (New England BiolabsGmbH). The product was PCR-purified (QIAquick PCR-Purification Kit;Qiagen) before being ligated into the EcoRI- and XbaI-predigestedpASK-IBA7 plasmid. The mutant library was transformed into E. coliXL2Blue (Stratagene, Amsterdam) using the TSS method [Chung C T, NiemelaS L and Miller R H, 1989. One-step preparation of competent Escherichiacoli: Transformation and Storage of Bacterial Cells in the SameSolution. Proc. Natl. Acad. Sci. U.S.A. 86: 2172-2175.].

d) Expression of the Butynol Esterase Mutant Library:

Colonies on LB_(amp) plates (=LB medium according to Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989 plus 10 g/l agar and 100 μg/mampicillin) were picked using toothpicks and transferred to microtiterplates with 96 wells containing 150 μl of LB_(amp) medium. Afterculturing in a microplate shaker (Multitron II; Infors GmbH, Einsbach,Germany; 37° C., 900 rpm and 70% humidity), ˜5 μl of each culture weretransferred using system Duetz (Kühner, Birsfelden, Switzerland) toplates with 2 ml wells containing 550 μl of TB medium with 100 μg mlampicillin (Becton Dickinson GmbH). The clones were cultured in aMultitron II shaker (Infors GmbH; 30° C. and 500 rpm and 70% humidity)for 8 hours. Protein expression was induced by adding 50 μl TBcomprising 2.4 μg/ml anhydrotetracycline (IBA GmbH) to each well. Theclones were cultured for another 6 hours in a Multitron II shaker(Infors GmbH; 30° C. and 500 rpm) for 8 hours.

e) pH Indicator Assay (96-Well Format):

A 50 μl TB culture from each well was transferred to 96-well assayplates using a Multimek 96-channel automatic pipetter (Beckman Coulter,Krefeld, Germany). The hydrolytic reaction was started by adding 100 μlof substrate solution (25 mM NaH₂PO₄ buffer, pH 7.5, ionic strength 154mM, adjusted with NaCl, comprising 0.05% Triton X-100, 7.5 μg/mlfluorescein-sodium and 285 mM but-3-yn-2-yl butyrate) to each well ofthe assay plate, using a Multimek 96 (Beckman Coulter). The substratesolution was agitated vigorously for 5 min prior to use. The kinetics ofreduction of the fluorescence signal (extinction 485 nm, emission 520nm) was observed using Tecan Safire (Tecan GmbH, Crailsheim, Germany)for 10 min. The activity was determined from the initial slope. Afterthe screening of 930 clones, clone 8H1 was identified as an improvedmutant.

f) Second Butynol Esterase Mutant Library:

A second butynol esterase mutant library was generated under exactly thesame conditions as described above, except that 8H1 was used as parentalgene. The library was expressed and screened as above. After a screeningof 1116 clones, clone 8C9 was identified as improved mutant.

g) Saturation Mutagenesis in Amino Acid Positions 190 and 263:

A saturation mutagenesis of amino acid positions 190 and 263 was carriedout using the following modified QuikChange protocols (Stratagene). Thefollowing mutagenesis primers were used for amino acid position 190:

(SEQ ID NO: 15) 5′-GGCATCCCGATCATGNNNCTGCAAAGCCGCAAG-3′ (SEQ ID NO: 16)5′-CTTGCGGCTTTGCAGNNNCATGATCGGGATGCC-3′

The following mutagenesis primers were used for amino acid position 263:

(SEQ ID NO: 17) 5′-GGGCGCCAGGACGCGNNNCTCGATTTCCACAAG-3′ (SEQ ID NO: 18)5′-CTTGTGGAAATCGAGNNNCGCGTCCTGGCGCCC-3′

The 50 μl PCR mixture (95° C. 30 s; 1 cycle//95° C. 30 s, 55° C. 1 min,68° C. 4 min 45 s; 18 cycles) comprised a mixture of 20 μmol of eachprimer, 0.2 mM of each dNTP (Roche Diagnostics GmbH), 50 ng of plasmid8C9 (starting DNA) and 2.5 U of Pfu Turbo DNA polymerase (Stratagene).The PCR was followed by digesting methylated and hemi-methylatedparental DNA with 40 U of DpnI (New England Biolabs GmbH) at 37° C. for2 to 3 hours. The products were PCR-purified (QIAquick PCR-PurificationKit; Qiagen) and then transformed into E. coli XL2Blue (Stratagene)using the TSS method [Chung C T, loc. cit].

The two libraries were expressed and screened in a manner identical tothat described above. After a screening of 186 clones for each library,two clones were identified as improved mutants. These were referred toas Est190-1 B2 and Est263-2D6.

h) Protein Expression in Shaker Flasks

Overnight cultures were grown in 5 ml of LB_(amp) medium using aMultitron-II shaker (Infors GmbH; 37° C., 250 rpm) by picking a freshlytransformed colony. 100 ml of TB_(amp) medium were inoculated with 800μl of said overnight culture and cultured in a Multitron-II shaker(Infors GmbH; 37° C., 200 rpm). Once OD₆₀₀ values of from 0.5 to 0.6 hadbeen reached, protein expression was induced by adding 25 μl ofanhydrotetracycline (stock solution of 0.2 mg/ml in DMF). The culturewas grown for another 12 hours (Multitron-II shaker, Infors GmbH; 30°C., 200 rpm). The cells were removed by centrifugation at 3220 g and 4°C. for 30 min and the wet cell mass was determined. The cell pelletswere stored at −20° C. until further characterization.

i) Protein Characterization using pH Stat:

The hydrolytic activity of butynol esterase was measured using pH stat(716 DMS Titrino, Deutsche Metrohm GmbH & Co., Filderstadt, Germany).

The cell pellets were first resuspended in a suitable volume of sodiumphosphate buffer (25 mM sodium dihydrogen phosphate buffer, pH 7.5,ionic strength: 154 mM, adjusted with NaCl) in order to obtain a cellconcentration of 0.1 g of wet cell mass/ml. 20 ml of substrate solutioncomprising sodium phosphate buffer (25 mM NaH₂PO₄ buffer, pH 7.5, ionicstrength: 154 mM, adjusted with NaCl), 0.05% Triton X-100 and 350 mMbut-3-yn-2-yl butyrate were prepared. The substrate solution wasagitated vigorously for 5 min prior to use.

The pH of the substrate solution (20 ml) was adjusted to pH 7.5 with 1NNaOH prior to starting the hydrolytic reaction. The reaction was startedby adding 0.01 g of wet cell mass. A pH of 7.5 was maintained bytitration with 0.1N NaOH during bioconversion. The hydrolytic activitywas correlated to the rate of titration of NaOH (volume, added per unittime).

The results are depicted in attached FIG. 4. The enzymes and mutantslisted in the following table were assayed. Information regarding theparticular mutation in the amino acid sequence and nucleic acid sequencecan likewise be found in said table.

Amino acid position Clones 16 190 263 pASK-IBA7- ctg (L) att (I) atc (I)Esterase 8H1 ccg (P) act (T) atc (I) 8C9 ccg (P) act (T) gtc (V)Est190-1B2 ccg (P) cgc (R) gtc (V) Est 263-2D6 ccg (P) act (T) gtt (V)

What is claimed is:
 1. An isolated mutant protein having esteraseactivity, wherein said isolated mutant protein consists of an amino acidsequence which is at least 95% identical to the amino acid sequence ofSEQ ID NO: 8 and differs from the amino acid sequence of SEQ ID NO: 8 bya mutation in an amino acid position corresponding to any one ofresidues 12-20 and 185-195 of the amino acid sequence of SEQ ID NO: 8,wherein said isolated mutant protein cleaves the ester but-3-yn-2-ylbutyrate, and wherein the isolated mutant protein has a calculatedmolecular weight of 38 to 34 kDa.
 2. An isolated esterase mutant havingesterase activity, wherein said isolated esterase mutant differs fromthe amino acid sequence of SEQ ID NO: 2 or 8 by a first mutation in anyof amino acid positions corresponding to residues 12-20 of the aminoacid sequence of SEQ ID NO: 2 or 8 and a second mutation in any of aminoacid positions corresponding to residues 185-195 of the amino acidsequence of SEQ ID NO: 2 or 8, and wherein said isolated esterase mutanthas at least 95% amino acid sequence identity to the amino acid sequenceof SEQ ID NO: 2 or
 8. 3. The isolated esterase mutant according to claim2, wherein the first mutation is at the amino acid positioncorresponding to residue 16 of the amino acid sequence of SEQ ID NO: 2or 8 and the second mutation is at the amino acid position correspondingto residue 190 of the amino acid sequence of SEQ ID NO: 2 or
 8. 4. Theisolated esterase mutant according to claim 3, wherein the first and/orsecond mutation is selected from the group consisting of Leu16Pro,Ile190Thr, and Ile190Arg.
 5. The isolated mutant protein according toclaim 1, wherein said isolated mutant protein catalyzes at least one ofthe following reactions: a) enantioselective hydrolysis of opticallyactive esters of the formula IR¹—COO—R²  (I), in which R¹ is a straight-chain or branched, optionallymono- or polysubstituted C₁-C₁₀-alkyl, C₂-C₁₀-alkenyl, C₂-C₁₀-alkynyl,and R² is a straight-chain or branched, optionally mono- orpolysubstituted C₁-C₁₀-alkenyl, C₂-C₁₀-alkynyl, C₇-C₁₅-aralkyl or amono- or polynuclear, optionally mono- or polysubstituted aromaticradical, R¹ and/or R² comprise at least one asymmetric carbon; and b)enantioselective transesterification of an ester of the formula I withan optically active alcohol of the formula IIR²—OH  (II), in which R² is a straight-chain or branched, optionallymono- or polysubstituted. C₁-C₁₀-alkyl, C₂-C₁₀-alkenyl, C₂-C₁₀-alkynyl,C₇-C₁₅-aralkyl or a mono- or polynuclear, optionally mono- orpolysubstituted aromatic radical and comprises at least one asymmetriccarbon.
 6. A process for enantioselective ester hydrolysis using theisolated mutant protein according to claim 1, which process comprises a)contacting said isolated mutant protein with a stereoisomer mixture ofan optically active ester of the formula R¹—COO—R² (formula I) in areaction medium to enantioselectively hydrolyze the stereoisomer mixtureand produce optically active compounds; and b) obtaining the opticallyactive compounds from the reaction medium, wherein R¹ is astraight-chain or branched, optionally mono- or polysubstitutedC₁-C₁₀-alkyl, C₂-C₁₀-alkenyl, C₂-C₁₀-alkynyl, and R² is a straight-chainor branched, optionally mono- or polysubstituted C₁-C₁₀-alkyl,C₂-C₁₀-alkenyl, C₂-C₁₀-alkynyl, C₇-C₁₅-aralkyl or a mono- orpolynuclear, optionally mono- or polysubstituted aromatic radical, andR¹ and/or R² comprise at least one asymmetric carbon.
 7. A process forenantioselective transesterification, which comprises a) contacting astereoisomer mixture of an optically active alcohol of the formula R²—OH(formula II) with an ester of the formula R¹—COO—R² (formula I) in thepresence of the isolated mutant protein according to claim 1 in areaction medium and obtaining the unreacted alcohol stereoisomer fromthe reaction medium; or b) contacting a stereoisomer mixture of anoptically active ester of the formula I with an alcohol of the formulaII in the presence of said isolated mutant protein in a reaction mediumand obtaining a stereoisomer of the optically active alcohol from thereaction medium, wherein R¹ of formula I is a straight-chain orbranched, optionally mono- or polysubstituted C₁-C₁₀-alkyl,C₂-C₁₀-alkenyl, C₂-C₁₀-alkynyl, and R² of formula I is a straight-chainor branched, optionally mono- or polysubstituted C₁-C₁₀-alkyl,C₂-C₁₀-alkenyl, C₂-C₁₀-alkynyl, C₇-C₁₅-aralkyl or a mono- orpolynuclear, optionally mono- or polysubstituted aromatic radical, andR¹ and/or R² comprise at least one asymmetric carbon, and wherein R² offormula II is a straight-chain or branched, optionally mono- orpolysubstituted C₁-C₁₀-alkyl, C₂-C₁₀-alkenyl, C₂-C₁₀-alkynyl,C₇-C₁₅-aralkyl or a mono- or polynuclear, optionally mono- orpolysubstituted aromatic radical and comprises at least one asymmetriccarbon.
 8. The process according to claim 7, wherein the ester is avinyl ester.
 9. The process according to claim 6, wherein the reactionmedium comprises an organic solvent.
 10. The process according to claim7, wherein the reaction medium comprises an organic solvent.
 11. Anisolated esterase mutant having esterase activity, wherein said isolatedesterase mutant differs from the amino acid sequence of SEQ ID NO: 2 or8 by a first mutation in any of amino acid positions corresponding toresidues 12-20 of the amino acid sequence of SEQ ID NO: 2 or 8, a secondmutation in any of amino acid positions corresponding to residues185-195 of the amino acid sequence of SEQ ID NO: 2 or 8, and a thirdmutation in any of amino acid positions corresponding to residues258-268 of the amino acid sequence of SEQ ID NO: 2 or 8, and whereinsaid isolated esterase mutant has at least 95% amino acid sequenceidentity to the amino acid sequence of SEQ ID NO: 2 or
 8. 12. Theisolated esterase mutant according to claim 11, wherein the thirdmutation is at the amino acid position corresponding to residue 263 ofthe amino acid sequence of SEQ ID NO: 2 or
 8. 13. The isolated mutantprotein according to claim 1, wherein the isolated mutant protein has atotal length of 335 amino acids and consists of an amino acid sequencewhich is at least 95% identical to the amino acid sequence of SEQ ID NO:8.
 14. The isolated esterase mutant according to claim 11, wherein thefirst mutation is at the amino acid position corresponding to residue 16of the amino acid sequence of SEQ ID NO: 2 or 8, the second mutation isat the amino acid position corresponding to residue 190 of the aminoacid sequence of SEQ ID NO: 2 or 8, and the third mutation is at theamino acid position corresponding to residue 263 of SEQ ID NO: 2 or 8.